HLA class I antigen downregulation in primary and metastatic locoregional melanoma lesions is significantly, although not absolutely, associated with poor prognosis. It is our working hypothesis that these findings reflect the lack of complete correlation of the level of the HLA class I restricting allospecificity and melanoma associated antigen (MAA) with that of HLA class I antigen-MAA peptide complexes on melanoma cells because of the role played by components of the antigen processing machinery. To test this hypothesis we will develop probes, i.e. scFv fragments and/or mAb, to measure HLA class I antigen-MAA peptide complexes on melanoma cells. Utilizing these probes we will test the influence of the level of the restricting HLA class I allospecificity, selected MAA, components of the antigen processing machinery and chaperone molecules on the level of HLA class I antigen-MAA peptide complexes on melanoma cells. Analysis of the expression of LMP2 and LMP7 in the proteasome will be emphasized since a recent study has unexpectedly shown that LMP2 and LMP7 expression in the proteasome inhibits MART-1 presentation to CTL. The functional significance of HLA class I antigen-MAA complex expression on melanoma cells will be investigated by correlating its level with the extent of HLA class I antigen restricted, MAA specific CU activation. The clinical significance of HLA class I antigen-MAA complex expression in melanoma lesions will be investigated by correlating its level with the clinical course of the disease and with the response to T cell-based immunotherapy. These hypotheses will be initially tested with HLA-A*0201 antigen and gp100 derived peptide 209-2 17, since the high frequency of expression of these markers facilitates the accrual of lesions to the study. Subsequent studies will extend these investigations to other HLA class I allospecific4ties and MAA. The outlined studies are expected to contribute to our understanding of the variables which regulate the expression of HLA class I antigen-MAA peptide complexes on melanoma cells and of the functional and clinical significance of the level of these complexes in melanoma lesions. Furthermore, the outlined studies are expected to yield methodology and reagents which will be very useful to screen and monitor patients with malignant diseases to be treated with T cell-based immunotherapy.